Project Summary/Abstract Gene expression is extensively controlled at the level of transcription initiation by processes that orchestrate assembly of the multi-subunit RNA polymerase II (Pol II) preinitiation complex (PIC) at promoters. Prevailing models derived from in vitro observations posit a defined pathway for assembly of the PIC and formation of stable promoter-bound complexes that facilitate transcription reinitiation and perpetuate the activated state. However, pathways for PIC assembly in vivo are not well understood, and emerging evidence suggests much more dynamic interactions between PIC components and promoters than have been observed in vitro. The broad, long-term objectives of the proposed project are to elucidate pathways and mechanisms responsible for assembly and activity of PICs in vivo. To address this goal, in this proposal we will apply novel methods that we developed in the prior period for measuring chromatin-binding dynamics in budding yeast cells. Our multi-disciplinary approaches provide quantitative estimates of transcription factor (TF) binding kinetics to single-copy loci including on- and off-rates as well as fractional occupancies of a DNA site by a TF in a cell population. In Aim 1, we will measure both chromatin binding dynamics of critical transcriptional components and RNA synthesis dynamics at a model activated gene, and mutational analyses will be used to determine the relationships between them. In Aim 2, we will measure chromatin-binding dynamics of key components of the PIC, genome-wide, and determine the relationships between kinetic behavior and gene regulatory properties, RNA synthesis rates, chromatin environment, and other properties. This landscape of kinetic properties will reveal the in vivo scope and scale of PIC assembly processes as they unfold in cells. Our prior work indicates that the dynamic properties of a PIC component called the TATA-binding protein (TBP) are controlled by an essential ATPase called Mot1. In Aim 3, we will use combined approaches to test specific models for Mot1's function in gene activation, and in so doing shed light on how regulation of chromatin binding dynamics can impact gene expression on a global scale. There is widespread allelic variation in TF binding sites, and such variation, as well as alterations in TF expression levels, can lead to differences in chromatin occupancy that contribute to numerous and prevalent human diseases, including obesity, cardiovascular disease, mental illnesses, and cancer. TFs themselves have been largely refractory to pharmacologic intervention; instead, we propose that a quantitative understanding of TF binding and PIC assembly dynamics in vivo will identify kinetic bottlenecks that will provide a foundation for ultimately developing entirely new approaches to treat transcriptional defects in human development and disease.